Ribonucleases (RNases) are a family of nucleases involved in the processing and degradation of RNAs (e.g., mRNAs and noncoding RNAs), which are critical for life cycles and cellular defense against infections. Change in RNase activity may be an indicator of human diseases (e.g., inflammation and cancer) and/or disease status. In addition, determination of RNase contamination is essential for RNA research and discovery.
Existing methods for detecting RNase activity include:
1. UV-based assays, which is less selective and thus unable to provide an accurate quantification.
2. Radioactivity-based assays, which requires the production and use of radioactive isotope-labeled RNA substrates.
3. Fluorescence-based assays, which requires the production and use of RNA substrates labeled with fluorophores.
The traditional Kunitz RNase activity assay is based upon the ultraviolet absorbance of label-free nucleic acids or degraded nucleosides, which is less selective, sensitive and accurate. Recent and current RNase activity assays including those commercially-available kits rely on isotope- or fluorophore-labeled RNAs (or antibodies), and thus offer greater sensitivities to determine very low levels of RNase activities (or indicate RNase protein levels). However, labeling methods are limited to the access to inexpensive, large quantities of labeled RNA substrates (e.g., micrograms), whereas biological samples such as human sera are comprised of high levels of RNase activities. Without extensive dilutions (e.g., 1:1,000) of the serum sample that would inevitably affect the RNase activity assay including linear range and accuracy, larger quantities (e.g., >10 μg) of labeled synthetic RNA agents are needed for a direct detection of serum RNase activity using labeling methods. Therefore, direct quantification of RNase activity in serum and other biological samples using labeling assays is costly and a more efficient method is warranted.